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Dissecting the Regulatory Mechanism of the Mammalian Microtubule-Severing Protein Katanin in Mitosis

  • Author(s): Cheung, Chingnam
  • Advisor(s): Torres, Jorge Z
  • et al.
Abstract

The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized.

To better understand the human Katanins and more broadly the mechanisms controlling mammalian microtubule-severing, we first analyzed the human Katanin interactome (Katan-ome) through biochemical tandem affinity purifications and mass proteomic analyses. This revealed that all Katanin subunits could reciprocally co-purify with each other, with the exception of KATNAL2 that only purified with KATNA1. These interactions were verified by in vitro and in cell reciprocal co-immunoprecipitations, with the exception of the KATNAL2-KATNA1 interaction. Analysis of the cell cycle subcellular localization of all Katanin subunits showed that KATNA1, KATNAL1 and KATNB1 localized to the cytoplasm during interphase and to the spindle poles during mitosis. Surprisingly, KATNBL1 localized to the nucleus during interphase (dependent on an N-terminal nuclear localization signal) and to the spindle poles during mitosis. In cell microtubule-severing assays with each A subunit revealed that unlike KATNA1 and KATNAL1, which showed microtubule-severing activity, KATNAL2 lacked detectable microtubule-severing activity. Interestingly, in vitro microtubule-severing assays showed that full-length KATNBL1 regulated KATNAL1 microtubule-severing activity in a concentration dependent manner. Furthermore, KATNB1 was able to compete the KATNBL1-KATNA1 and KATNBL1-KATNAL1 interactions.

These results indicate that KATNBL1 is a regulator of microtubule-severing activity and that it cooperates/competes with KATNB1 for binding to KATNA1 and KATNAL1 and that KATNB1 and KATNBL1 together regulate Katanin A subunit microtubule-severing activity.

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